STABILITY INDICATING METHOD DEVELOPMENT AND VALIDATION FOR SIMULTANEOUS ESTIMATION OF EMTRICITABINE AND TENOFOVIR DISOPROXIL FUMERATE IN BULK AND TABLET DOSAGE FORM BY RP HPLC

STABILITY INDICATING METHOD DEVELOPMENT AND

VALIDATION FOR SIMULTANEOUS ESTIMATION OF

EMTRICITABINE AND TENOFOVIR DISOPROXIL FUMERATE IN

BULK AND TABLET DOSAGE FORM BY RP-HPLC

Modugula Jyothsna* and Podili Venkata Seshagiri

Department of Pharmaceutical Analysis SIMS College of Pharmacy, Guntur-522 001,

Andhra Pradesh, India.

ABSTRACT

An accurate, precise and simple stability indicating Chromatographic

method for development and validation for the simultaneous estimation

of Emtricitabine and tenofovir Disoproxil Fumerate in its

pharmaceutical dosage form by HPLC was carried out by using ACE

C18 (150 mmx4.6 mm i.d, 5 μ) column with gradient program, mobile

phase A: 0.01M Citrate buffer pH 4.0 with Ammonia & mobile phase

B: ACN , Flow rate 1.2 ml/min., Injection volume 20 μl, UV detection

was performed by using wavelength at 270 nm.The method was linear

over the concentration range of 20 µg/mL to 70 µg/mL for

Emtricitabine (r2- 0.9997) and 25 µg/mL to 100 µg/mL for Tenofovir

disoproxil fumerate (r2- 0.9996) with limits of detection and

quantification of 0.112 μg/ml & 0.346 μg/ml for Emtricitabine and 0.192 μg/ml & 0.585

μg/ml for tenofovir Disoproxil Fumerate. Forced degradation study was carried out according

to ICH guidelines in Acid Degradation, Base Degradation, Oxidative Degradation, Thermal

Degradation conditions and the method was specific. A study to establish the stability of

standard and test preparations on bench top was conducted and the solutions were stable for

24 hours.

KEYWORDS: Emtricitabine, Tenofovir Disoproxil Fumerate, RP-HPLC, ICH Guidelines.

INTRODUCTION

Emtricitabine works by inhibiting reverse transcriptase, the enzyme that copies HIV RNA

into new viral DNA. Emtricitabine is a synthetic nucleoside analogue of cytidine. It is

Volume 8, Issue 5, 1028-1046. Research Article ISSN 2277– 7105

Article Received on 06 Feb. 2019,

Revised on 26 Feb. 2019, Accepted on 21 March 2019

DOI: 10.20959/wjpr20195-14618

*Corresponding Author

Prof. Jyothsna Modugula

Department of

Pharmaceutical Analysis SIMS College of Pharmacy, Guntur-522 001, Andhra Pradesh, India.

phosphorylated by cellular enzymes to form Emtricitabine 5'-triphosphate, which is

responsible for the inhibition of HIV-1 reverse transcriptase. It competes with the natural

substrate deoxycytidine 5'-triphosphate and incorporates into nascent viral DNA, resulting in

early chain termination. Therefore Emtricitabine inhibits the activity of HIV-1 reverse

transcriptase (RT) both by competing with the natural substrate deoxycytidine 5'-triphosphate

and by its incorporation into viral DNA. By inhibiting HIV-1 reverse transcriptase,

emtricitabine can help to lower the amount of HIV or "viral load", in a patient's body and can

indirectly increase the number of immune system cells (called T cells or CD4+ T-cells). Both

of these changes are associated with healthier immune systems and decreased likelihood of

serious illness. Tenofovir inhibits the activity of HIV reverse transcriptase by competing with

the natural substrate deoxyadenosine 5’-triphosphate and, after incorporation into DNA, by

DNA chain termination. Specifically, the drugs are analogues of the naturally occurring

deoxynucleotides needed to synthesize the viral DNA and they compete with the natural

deoxynucleotides for incorporation into the growing viral DNA chain. However, unlike the

natural deoxynucleotides substrates, NRTIs and NtRTIs (nucleoside/tide reverse transcriptase

inhibitors) lack a 3'-hydroxyl group on the deoxyribose moiety. As a result, following

incorporation of an NRTI or an NtRTI, the next incoming deoxynucleotide cannot form the

next 5'-3' phosphodiester bond needed to extend the DNA chain. Thus, when an NRTI or

NtRTI is incorporated, viral DNA synthesis is halted, a process known as chain termination.

All NRTIs and NtRTIs are classified as competitive substrate inhibitors.

MATERIALS AND METHODS

Emtricitabine and Tenofovir disoproxil fumerate API was obtained as a gift sample from

MYLAN,Bollaram, Hyderabad.

Table.1: Instruments Used.

S. No. Name of Instruments

1 Waters HPLC 2695 with UV detector

2 ACE C18 column 150 X 4.6, 5µ

3 Thermo scientific Finn Pipette

4 Inolab WTW series pH meter

5 Afroset Electronic Balance FX -400

6 Elma S 300H Ultra sonicator

7 Spectra lab water bath shaker

8 Hermle Z 323 centrifuge

Table.2: Chemicals Used.

S. No. Name Grade Make

1 Citric acid monohydrate Analytical Reagent Merck

2 Ammonia HPLC Merck

3 ACN HPLC Merck

4 Hydrochloric acid HPLC Merck

5 Water HPLC Milli Q (Purificationsystem)

Selection of Wavelength: The sensitivity of method that uses UV detector depends upon the

proper selection of wavelength is that gives maximum absorbance and good response for the

given candidate drug. In setting up the conditions for development of the assay method, the

choice of the detection wavelength was based on the scanned absorption spectrum for

Emtricitabine and Tenofovir disproxil fumerate. The UV-spectrum of Emtricitabine and

Tenofovir disproxil fumerate was separately scanned in the wavelength range of 200-400 nm

against blank. After correlation of the both spectrums 270nm wavelength was selected for the

analysis.[1-6]

3.211 Emtricitabine 6.271 Tenofovir 208.0

261.0 214.0

238.0 281.0

AU

0.00 0.05 0.10 0.15 0.20 0.25 0.30 0.35 0.40 0.45

nm

200.00 220.00 240.00 260.00 280.00 300.00 320.00 340.00 360.00 380.00 400.00

Figure. 1: Overlay UV spectrum of EMT and TDF.

Optimized Chromatographic Conditions

Column : ACE C18 (150 mmx4.6 mm i.d, 5 μ)

Flow rate : 1.2 mL/min

Selected wave length : 270nm

Program : Gradient

Detector : UV Detector/ PDA

Mobile phase A : 0.01M Citrate buffer pH 4.0 with Ammonia

Injection volume : 20 μL

Temperature : 30ºC

Time (min) Mobile phase-A Mobile phase-B

0 70 30

4 20 80

7 20 80

8 70 30

12 70 30

Preparation of 0.01M Hydrochloric Acid (Diluent): About 8.5mL of concentrated

hydrochloric acid was diluted to 1000 mL with water in a volumetric flask and mixed well.

100 mL of this solution was further diluted to 1000 mL with water and mixed.

Preparation of 0.01M Citrate Buffer (Mobile Phase A): Accurately 2 gm of citric acid

monohydrate was weighed and transferred in to a 1000 mL standard flask and diluted to 1000

mL with water. The pH was adjusted to 4.0 with ammonia, filtered and degassed.

Preparation of Mobile Phase B

ACN was used as mobile phase B.

Preparation of Standard Stock Solution: Accurately 40 mg of Emtricitabine and 60mg of

Tenofovir disoproxil fumerate working standard were transfered into a 100mL volumetric

flask. 50 mL diluent was added and sonicated to dissolve and made up to volume with

diluent.

Preparation of Standard Solution: About 5 mL of the standard stock solution was pipetted

out into a 50mL volumetric flask and diluted up to the volume with diluent and filtered

through 0.45 PVDF filter.

Test Preparation: Accurately weighed amount of the powder equivalent to 200mg of

Emtricitabine and 300 mg of Tenofovir disoproxil fumerate were transfered into a 500mL

volumetric flask. 400mL of diluent was added and sonicated for 45min with intermediate

shaking. It is allowed to cool at room temperature, diluted to volume with diluent and mixed

well. A portion of the above solution was centrifuged in a centrifuge tube with cap at 5000

rpm for 10 min. About 5.0 mL of the above solution was pipetted into 50mL volumetric flask

and diluted to volume with diluent and mixed well. The solution was filtered through 0.45µ

Method Validation

System suitability[7-9]

About 20µL of blank, standard solution (six times) was injected, chromatogram was recorded

and peak response was measured.

The tailing factor for the Emtricitabine and Tenofovir disoproxil fumerate peak should be not

more than 2.0 from the chromatogram of standard solution.

The Relative standard deviation of Emtricitabine and Tenofovir disoproxil fumerate peak area

from six replicate injections of standard solution should be not more than 2.0%.

Table. 3: Data for System Suitability.

System suitability parameters Observed values Acceptance

criteria Emtricitabine Tenofovir DF

The USP plate count from standard chromatogram 13438 48847 NLT 3000

The Tailing factor from standard chromatogram 1.06 1.05 NMT 2

% RSD from 6 replicate injections of standard solution 0.14 0.15 NMT 2

e

m

tr

ici

ta

b

in

e

3

.1

5

2 te

n

o

fo

v

ir

6

.0

8

4

AU

0.00 0.05 0.10 0.15 0.20 0.25 0.30 0.35 0.40

Minutes

2.00 4.00 6.00 8.00 10.00 12.00

Figure. 2: System Suitability Chromatogram for Standard.

System Precision

Procedure

System precision was performed by injecting six replicate injections of Emtricitabine and

Tenofovir disoproxil fumerate (40 ppm of Emtricitabine and 60ppm of Tenofovir disoproxil

Table. 4: Data for System precision for Emtricitabine.

Injection Number Emtricitabine Peak area

1 1276471

2 1255856

3 1254000

4 1252648

5 1256997

6 1255857

Average 1256313.167

SD 1643.222373

%RSD 0.14

For Tenofovir disoproxil fumerate

Injection number Tenofovir DF Peak area

1 1609813

2 1610262

3 1606962

4 1604478

5 1610537

6 1606525

Average 1608096.167

SD 2467.470236

%RSD 0.15

Precision of Test Method

Table 5: Data for Method precision for Emtricitabine.

S. No. Area % Assay

1. 1146433.0 99.1

2. 1163281.5 100.7

3. 1136656.5 98.3

4. 1148538.5 99.2

5. 1136979.0 98.5

6. 1151405.0 99.5

Mean 99.2

% RSD 0.9

For Tenofovir disoproxil fumerate

S. No. Area % Assay

1. 1608364.5 99.6

2. 1600735.0 99.2

3. 1596586.5 98.9

4. 1602959.0 99.2

5. 1576688.5 97.9

6. 1599553.0 99.0

Mean 99.0

Intermediate Precision

The ruggedness of method was demonstrated by conducting the precision study on different

HPLC system and different column of same make on different day. Assay was performed for

six individual test preparations of 200/300mg strengths as per test method.

Table. 6: Data for Intermediate Precision (Column to Column, System to System

variation).

System suitability parameters

Observed value

Acceptance criteria Column 1 &

system 1

Column 2 & system 2

The Tailing factor for Emtricitabine peak 1.06 1.02 NMT 2.0

The Tailing factor for Tenofovir DF peak 1.05 1.01 NMT 2.0

% Relative standard deviation for Emtricitabine peak 0.14 0.24 NMT 2.0

% Relative standard deviation for Tenofovir DF peak 0.15 0.14 NMT2.0

*(n = 6)

Table. 7: Datafor Intermediate Precision for EMT and TDF.

S. No.

% Assay

Emtricitabine Tenofovir DF

Column–1 and system 1

Column–2 and system-2

Column–1 and system-1

Column–2 and system-2

1 99.1 100.6 99.6 100.3

2 100.7 98.4 99.2 98.2

3 98.3 100.5 98.9 100.2

4 99.2 100.8 99.2 100.6

5 98.5 98.5 97.9 98.3

6 99.5 98.7 99.0 98.3

Average 99.2 99.6 99.0 99.3

% RSD 0.9 1.2 0.6 1.2

Linearity

Linearity of detector response was established by plotting a graph between concentrations

versus peak area. A series of solutions Emtricitabine and Tenofovir disoproxil fumerate

standard was prepared in the concentration ranging from 20 µg/mL to 70 µg/mL for

Emtricitabine and 25 µg/mL to 100 µg/mL for Tenofovir disoproxil fumerate and analyzed as

per test method. A graph was plotted with concentration in µg/mL on X- axis versus response

Table. 8: Data for Linearity for Emtricitabine.

S. No % Level Concentration (µg/mL) Peak Area

1 40 20.032 462372

2 60 28.048 713211

3 80 35.064 948758

4 100 48.079 1165361

5 120 57.095 1405071

6 160 70.127 1892006

For Tenofovir disoproxil fumerate

S. No % Level Concentration (µg/mL) Peak Area

1 40 25.968 639745

2 60 40.952 978324

3 80 57.936 1306936

4 100 71.92 1592068

5 120 86.904 1917759

6 160 100.872 2572312

Table. 9: Data for Linearity Parameters for EMT and TDF.

S. No Parameter Results

EMT TDF

1 Linearity Range 20- 70 µg/mL 25- 100 µg/mL

2 Co-efficient of correlation(r2) 0.9997 0.9996

3 Slope 29483 26635

4 Intercept 1845.6 14314.9

5 Residual sum of squares 413931014.4 903880385.9

6 Standard Error for slope 262.72 259.68

7 Standard Error for y intercept 10669 15766

8 LOD(µg/mL) 0.112 0.192

9 LOQ(µg/mL) 0.346 0.585

L inearity g raph for E mtric itabine

y = 29483x - 1845.6 R ² = 0.9997

0 500000 1000000 1500000 2000000

0 10 20 30 40 50 60 70

C onc in P P M

A

re

a

L inearity g raph for tenofovir

y = 26658x + 14314.9 R ² = 0.9996

0 500000 1000000 1500000 2000000 2500000 3000000

0 20 40 60 80 100 120

C onc in P P M

A

re

a

Figure. 4: Calibration Plot for Tenofovir disoproxil fumerate.

Accuracy: The accuracy was confirmed by recovery studies by adding known amount of

placebo to the pure API of Emtricitabine and Tenofovir disoproxil fumerate from about 50%

to 150% of the initial assay concentration. Sample solutions was prepared in triplicate for

each level and analyzed as per test method.

Table 10: Data for Accuracy.

For Emtricitabine

Sample No. % Spike level mg

added

mg

found % Recovery

Mean % Recovery

% RSD

1 50 99.3 99.95 100.7

101.1 0.7

2 50 99.8 100.36 100.6

3 50 99.6 101.46 101.9

1 100 197.61 199.89 101.2

100.6 0.6

2 100 197.51 198.55 100.5

3 100 197.11 197.33 100.1

1 150 296.71 296.01 99.8

99.8 0.4

2 150 297.11 295.32 99.4

3 150 297.31 297.73 100.1

For Tenofovir disoproxil fumerate

Sample No.

% Spike level

mg added

mg found

% Recovery

Mean % Recovery

% RSD

1 50 149.35 148.97 99.7

99.7 0.8

2 50 149.15 147.58 98.9

3 50 149.15 149.76 100.4

1 100 296.71 293.58 98.9

99.4 0.8

2 100 297.21 294.18 99.0

3 100 296.81 298.11 100.4

1 150 446.16 443.26 99.4

99.6 0.3

2 150 445.96 443.22 99.4

Interference from Degradation Products

A study was conducted to demonstrate the effective separation of degradants from

Emtricitabine and Tenofovir disoproxil fumerate peak in Assay method. Separate portions of

drug product and placebo were exposed to the following stress conditions to induce

degradation.[12-15]

1) Treated with 0.1N HCL solution.

2) Treated with 0.1 NaOH solution.

3) Treated with 3% H2O2 solution for about 5 minutes on bench top.

4) Exposed to heat for about 24 hrs at 105ºC.

The chromatograms of the stressed samples were evaluated for peak purity of Emtricitabine

and Tenofovir disoproxil fumerate.

5) Exposed to humidity at 25ºC/90%RH for about 48 hrs.

2 .0 8 7 2 .2 3 8 E M T R IC IT A B IN E 3 .1 8 9 4 .5 5 7 T E N O F O V IR 6 .1 3 4 AU 0.00 0.05 0.10 0.15 0.20 0.25 Minutes

2.00 4.00 6.00 8.00 10.00 12.00

Figure. 5: Chromatogram for Acid Degradation Sample.

2 .2 4 2 2 .4 3 7 E M T R IC IT A B IN E 3 .1 9 0 3 .3 8 3 4 .5 5 8 5 .7 6 0 T E N O F O V IR 6 .1 3 0 AU 0.00 0.05 0.10 0.15 0.20 Minutes

2.00 4.00 6.00 8.00 10.00 12.00

1 .7 3 4 2 .0 8 9 E M T R IC IT A B IN E 3 .1 8 6 4 .1 0 2 4 .5 5 6 T E N O F O V IR 6 .1 2 9 AU 0.00 0.10 0.20 0.30 Minutes

2.00 4.00 6.00 8.00 10.00 12.00

Figure. 7: Chromatogram for Peroxide Degradation Sample.

1 .5 5 8 S -O x id e Im p u ri ty - 1 .9 8 2 2 .2 3 1 E m tr ic it a b in e 3 .2 2 6 3 .4 5 1 M o n o e st e r Im p u ri ty - 4 .6 7 1 5 .4 4 4 5 .7 7 6 T e n o fo v ir 6 .2 7 0 6 .5 0 0 6 .9 1 0 8 .2 7 9 AU 0.00 0.05 0.10 0.15 0.20 0.25 0.30 0.35 Minutes

0.00 1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00 9.00 10.00 11.00 12.00

Figure. 8: Chromatogram for Thermal Degradation Sample.

1 .5 5 8 S -O x id e Im p u ri ty - 1 .9 7 8 E m tr ic it a b in e 3 .2 1 4 3 .4 3 9 M o n o e st e r Im p u ri ty - 4 .6 7 1 T e n o fo v ir 6 .2 6 9 6 .4 9 0 8 .2 8 0 AU 0.00 0.05 0.10 0.15 0.20 0.25 0.30 0.35 Minutes

2.00 4.00 6.00 8.00 10.00 12.00

Table 11: Data for Emtricitabine Degradation.

Table. 12: Data for TDF Degradation.

Stability of Solutions

Bench Top Stability of Standard Preparation and Test Preparations

A study to establish the stability of standard and test preparations on bench top was

conducted for 24 hours. The assay of standard and test preparations was estimated against

freshly prepared standard at initial and after 24 hours.

Table. 13: Data for Bench Top Stability for Standard and Test Preparations For

Emtricitabine.

Time (Hrs)

% Assay of standard preparation

Difference from initial

% Assay of test

preparation Difference from initial

Test - 1 Test - 2 Test - 1 Test – 2

Initial 98 NA 100.7 98.3 NA NA

24 96.8 1.2 100.5 98.2 0.2 0.1

For Tenofovir disoproxil fumerate

Time (Hrs)

% Assay of standard preparation

Difference from initial

% Assay of test

preparation Difference from initial

Test - 1 Test - 2 Test - 1 Test – 2

Initial 97.8 NA 99.2 98.2 NA NA

24 96.8 1.0 100.2 99.6 1.0 1.4

NA- Not Applicable

Acceptance Criteria

The % assay results of standard and test solutions should not deviate by ±2.0 from that of

initial.

Name Retention Time Area % Area

Acid 3.189 1073283 40.36

Base 3.190 1068103 39.51

Oxidation 3.186 1031224 38.08

Thermal 3.226 1098375 38.18

Humidity 3.253 1042165 38.12

Name Retention Time Area % Area

Acid 6.134 1138997 42.83

Base 6.130 723577 26.77

Oxidation 6.129 1490223 55.02

Thermal 6.270 1518599 52.79

Bench Top Stability of Mobile Phase

A study to establish stability of mobile phase on bench top was conducted for 5 days.

The system suitability parameters were evaluated as per the test method by using the same

mobile phase on different days (initial, after 1 day, 3 days and 5 days).

Table 14: Data for Mobile Phase Stability on Bench top for Emtricitabine.

System

Suitability Parameters

Observed value

Acceptance Criteria (%RSD) At

Initial

After 1 day

At Day-3

At Day-5

The Tailing factor for Emtricitabine peak in Standard solution

1.06 1.07 1.06 1.04 NMT 2.0

%RSD of peak area of

Emtricitabine 0.14 0.14 0.08 0.03 NMT 2.0

USP plate count of

Emtricitabine peak 13438 12204 13411 11847 NLT 3000

For Tenofovir disoproxil fumerate

System

Suitability Parameters

Observed value

Acceptance Criteria (%RSD) At

Initial

After 1 day

At Day-3

At Day-5

The Tailing factor for

Tenofovir DFpeak 1.05 1.04 1.02 1.10 NMT 2.0

%RSD of peak area of

Tenofovir DF 0.15 0.27 0.07 0.03 NMT 2.0

USP plate count of

Tenofovir DF peak 48847 44742 45416 44478 NLT 3000

Robustness

Effect of Variation in Flow Rate

A study was conducted to determine the effect of variation in flow rate. The system

suitability parameters were evaluated at the flow rate of 0.9 mL/min and 1.1 mL/min.

Table. 15: Data for Effect of Variation in Flow Rate.

System Suitability Parameters Observed value Acceptance

Criteria

0.9mL/min 1.1 mL/min

The tailing factor for Emticitabine peak 1.07 1.06 NMT 2.0

The tailing factor for Tenofovir DF peak 1.06 1.03 NMT 2.0

% Relative standard deviation for Emtricitabine peak 0.26 0.09 NMT 2.0

Effect of Variation in Column Temperature

A study was conducted to determine the effect of variation in column oven temperature. The

system suitability parameters were evaluated at 25°C and 35°C column oven temperatures.

Table. 16: Data for Effect of Variation in Column Temperature.

System

Suitability Parameters

Observed value Acceptance

Criteria

25°C 35°C

The tailing factor for Emticitabine peak 1.07 1.08 NMT 2.0

The tailing factor for Tenofovir DFpeak 1.07 1.07 NMT 2.0

% Relative standard deviation for

Emtricitabine peak 0.11 0.05 NMT 2.0

% Relative standard deviation for

Tenofovir DF 0.11 0.06 NMT2.0

Effect of Variation in pH of Buffer in Mobile Phase

A study to establish the Effect of variation in pH of buffer in mobile phase was conducted.

Mobile phase was prepared with buffer having different pH between 4.3 and 4.7. System

suitability parameters were evaluated by using the above mobile phases.[16-18]

Table. 17: Data for Effect of Variation in pH of Buffer in Mobile Phase.

Filter Interference

A study to establish the suitability of filters was conducted by using two different filters

namely, 0.45µm PVDF filter (Mfg. by: M/s. Millipore) and Nylon 0.45 µm filters (Mfg. by:

M/s. Millipore). Test solutions prepared in duplicate was centrifuged and filtered through the

above different filters and analyzed as per test method. The difference in % Assay between

centrifuged and filtered test solutions was determined.

The standard solution prepared as per test method was filtered through the above different

filters and analyzed along with unfiltered standard solution.[13,14]

The similarity factor of Emtricitabine and Tenofovir disoproxil Fumerate in filtered standard

solutions against unfiltered standard solution was calculated.

System

Suitability Parameters

Observed value Acceptance

Criteria

pH 4.3 pH 4.7

The tailing factor for Emtricitabine peak 1.07 1.07 NMT 2.0

The tailing factor for Tenofovir DF peak 1.04 1.05 NMT 2.0

% Relative standard deviation for Emtricitabine peak 0.14 0.13 NMT 2.0

Filter validation

Table. 18: Description for Filters.

Filter Description Filters

Nylon PVDF

Manufacturers Name Millipore Millipore

Size 0.45µm 0.45µm

Table. 19: Results of Filter Interference for standard.

Sample Peak Area Similarity Factor

Emtricitabine Tenofovir DF

Centrifuged 1122852 1612386 NA

0.45µm PVDF 1120847 1607107 1.00

0.45µm NYLON 1119476 1604829 1.00

Table. 20: Results of Filter Interference for Test.

Sample Name

% Assay Difference between Centrifuged and filtered

sample

Centrifuged 0.45 µm

PVDF filter

0.45 µm

Nylon filter 0.45 µm PVDF filter 0.45 µm Nylon filter

Emtricitabine 97.3 97.1 97.3 0.2 0.0

Tenofovir 97.5 96.9 97.5 0.6 0.0

Acceptance Criteria: 1) The % assay of filtered test solutions should not deviate by ± 2.0

from that of centrifuged test solutions

RESULTS AND DISCUSSIONS

A wavelength of 270nm was selected as a detection wavelength for the estimation of

Emtricitabine and Tenofovir disoproxil fumerate in RP-HPLC system. A simple, precise and

accurate RP-HPLC method was developed for the analysis of Emtricitabine and Tenofovir

disoproxil fumerate in tablet dosage form using the mobile phase A consisting of 0.01M

citrate buffer (pH 4.0) and mobile phase B is 100% ACN. The flow rate was found to be

optimized at 1.2 mL/min. It reduced the usage of mobile phase. The system suitability

parameters like retention time, resolution, efficiency, capacity factor, tailing factor and %

RSD was found to be within the limits for the optimized chromatogram.

Method precision: The method precision study was conducted for the Emtricitabine and

Tenofovir disoproxil fumerate sample solutions. %RSD was found to be 0.9 for Emtricitabine

and 0.6 for Tenofovir disoproxil fumerate. Assay was performed to determine the purity of

was found to be 99.2% for Emtricitabine and 99.0% for Tenofovir disoproxil fumerate

respectively.

Intermediate precision: The intermediate precision (Ruggedness) was performed with two

different columns and different instrument but there is no change in retention time, system

suitability parameter and % content.

Linearity: It is evident that the responses for Emtricitabine and Tenofovir disoproxil

fumerate was found to be linear in the studied concentration ranges from 20µg/mL to

70µg/mL and 25µg/mL to 100µg/mL respectively and the correlation coefficient was found

to be r2=0.9997 and r2=0.9996 for Emtricitabine and Tenofovir disoproxil fumerate

respectively.

Accuracy: The accuracy studies was carried out at 3 levels (50%, 100% and 150%) to ensure

the accuracy of the method by adding known concentration of API to Placebo. The average

percentage recovery was found to be in the range of 100% Emtricitabine, 99.5% for

Tenofovir disoproxil fumerate respectively. Nearly 100% recovery showed that the method

was free from the interference of the excipients used in the formulation.

Specificity: The specificity was performed under stress conditions like acid, base, peroxide,

thermal and humidity was performed and observed the degradation of drugs. It was found to

be within the limits. From the above chromatograms of forced degradation studies, and their

purity plots, it can be inferred that peaks of the degradants was not interfere with the retention

time of the main peak of Emtricitabine and Tenofovir disoproxil fumerate.

Bench top stability: Bench top stability for standard, test and mobile phase was performed.

The difference in percentage assay of standard and test preparations between initial and after

24 hours is found to be within the limit. From the above study, it is concluded that the

standard and test preparations are stable for 24 hours on bench top. The mobile phase was

found to be clear and no haziness was observed during the stability period, it is concluded

that the mobile phase is stable for 5 days on bench top.

Robustness: The Robustness was performed by making deliberate changes in flow rate,

column temperature and pH of the buffer solution (mobile phase A). It shows that there is no

change in the retention time even after making deliberate change in the analytical procedure.

Filter validation: The filter validation was performed by using two different filters namely,

0.45µm PVDF filter (Mfg. by: M/s. Millipore) and Nylon 0.45 µm filters (Mfg. by: M/s.

Millipore). The similarity factor of Emtricitabine and Tenofovir disoproxil Fumerate in

filtered standard solutions against centrifuged standard solution is found to be within the

limit. From the above study, it is concluded that both the (PVDF and Nylon) filters are

suitable for standard and test solution filtration.

SUMMARY AND CONCLUSIONS

All the above parameters have shown that the developed method for the estimation of

emtricitabine and tenofovir disoproxil fumerate in bulk and tablet dosage form by rp-hplc

was successfully developed by using RP-HPLC and the method was validated as per the

ICH(Q2B)guidelines and the results have proved that the method is selective, precise,

accurate, stable and linear. Hence it was concluded that the developed method was found to

be applicable for routine quantitative analysis for the estimation of emtricitabine and

tenofovir disoproxil fumerate in bulk and tablet dosage form.

ACKNOWLEDGEMENT

The author would like to thankful the management of SIMS College of Pharmacy, Guntur,

for providing the necessary facilities to carry out this research work.

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